The flagellar pocket of Leishmania is the major site of exchange between the parasite and it's extracellular environment. All uptake of macromolecular nutrients and all secretion takes place through this region of the cell. We propose integrated biochemical and genetic approaches to the identification and functional characterization of several key constituents or activities involved in endocytosis through the flagellar pocket. The two specific issues addressed are:- 1. We have identified and cloned the gene encoding a protein (FLAP) homologous to the mammalian junction protein desmoplakin which localizes to the hemidesmosomal junction at the mouth of the flagellar pocket. We propose to define the function of the FLAP protein through manipulation of the gene. We will proceed through knockout of the FLAP gene and expression of dominant/negative forms of the different domains of the protein. The FLAP protein likely fulfills a similar role in all trypanosomatids and these studies will be extended to T. brucei because the inducible expression constructs for these parasites afford some distinct technical advantages. The latter approaches will enable experimentation even if the knockout phenotype is lethal. 2. We have shown that transferrin/toxin complexes efficiently kill Leishmania promastigotes and propose a genetic screen which will allow identification of the transferrin receptor gene(s). Parasites will be mutagenized and incubated in transferrin/toxin in medium containing an alternate iron source. Null mutants will be complemented with a cosmid library and selected in growth medium containing transferrin. The overlapping clones will be mapped to identify the ORFs encoding the tranferrin binding activity. These goals play to our strengths and established expertise in endocytosis and protein trafficking and will yield valuable information on the relative role of endocytosis and the flagellar pocket to the success of trypanosomatids in their different host environments.